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Millipore novablue
Novablue, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 99 stars, based on 14 article reviews
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novablue - by Bioz Stars, 2019-12
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Clone Assay:

Article Title: Design of pH Sensitive Binding Proteins from the Hyperthermophilic Sso7d Scaffold
Article Snippet: Cells from the final sort were plated on SDCAA plates and individual clones were picked for sequencing. .. The isolated DNA was further transformed into Novablue™ (E. coli ) cells (EMD Biosciences, San Diego, CA).

Article Title: Association of Mycobacterium Proteins with Lipid Droplets
Article Snippet: Going forward, this information may help in the development of novel therapeutics capable of disrupting key LD-protein interactions needed by M. tuberculosis for survival during nonreplicating persistence. .. Escherichia coli strains DH5α, Top10, (Thermo Fisher Scientific, Waltham, MA), and NovaBlue (MilliporeSigma, Burlington, MA) were used for cloning. .. Protein purification was carried out in E. coil strain BL21(DE3) containing plasmid pLysS.

Article Title: Probing the Substrate Specificity and Protein-Protein Interactions of the E. coli Fatty Acid Dehydratase, FabA
Article Snippet: The FabA gene was cloned into the pET28b expression vector and used as the DNA template for site-directed mutagenesis with the mutagenic primer listed in . .. Digested PCR products were transformed into NovaBlue (EMD Millipore).

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of His1 and its H-bonding Network
Article Snippet: The PCR product was gel purified (1% agarose) and treated with T4 DNA polymerase to create overhangs complementary to the Ligation Independent Cloning (LIC) vector pET30 Xa/LIC (Novagen™). .. The insert was annealed to the vector and NovaBlue (Novagen™) competent cells were transformed with the pET30 Xa/LIC plasmid containing the sodN mutant gene for selection.

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of Cys2 and Cys6
Article Snippet: The PCR product was gel purified (1% agarose) and treated with T4 DNA polymerase to create overhangs complementary to the Ligation Independent Cloning (LIC) vector pET30 Xa/LIC (Novagen™ ). .. The insert was annealed to the vector and NovaBlue (Novagen™ ) competent cells were transformed with the pET30 Xa/LIC plasmid containing the sodN mutant gene for selection.

Amplification:

Article Title: Concatenated Metallothionein as a Clonable Gold Label for Electron Microscopy
Article Snippet: The inserted MT gene was amplified from pET-3d-MT using the same forward primer as used for the pMAL-c2x-MT plasmid (above), and the reverse primer (5′-GTGACCACATGTCACAGCACGTGCACTTGTCC -3′), which contains an AflIII site at the MT-MT junction. .. The products of the ligation reactions for both constructs were transformed into Novablue (Novagen) E. coli cells.

Article Title: Characterization of a stalled complex on the β-barrel assembly machine
Article Snippet: NovaBlue (Novagen) cells were transformed with 1 μL of the ligation product and plated onto LB plates containing 50 μg/mL kanamycin. .. To construct pET9a FLAG 3 -BamA , a cassette containing the coding sequence of the FLAG3 tag was inserted into pET9a His-BamA to replace the original His6 tag.

Article Title: Characterization of a stalled complex on the β-barrel assembly machine
Article Snippet: Briefly, the entire pET9a His-BamA template was amplified by PCR (using primers 5′-A GAT CAT GAT ATC GAC TAT AAA GAC GAT GAT GAC AAA GAA GGG TTC GTA GTG AAA GAT-3′ and 5′-TCG ATA TCA TGA TCT TTG TAG TCG CCG TCG TGA TCT TTA TAA TCA GCA CCG TAT ACG GTG-3′) and the resulting PCR product mixture digested with DpnI for > 1 h at 37 °C. .. NovaBlue (Novagen) cells were transformed with 1 μL of digested PCR product and plated onto LB plates containing 50 μg/mL kanamycin.

Article Title: Characterization of a Novel Serine Protease Inhibitor Gene from a Marine Metagenome
Article Snippet: The PCR cycling consisted of denaturation steps (96 °C for 2 min), 30 cycles at 94 °C for 40 s, 58.5 °C for 30 s, and 72 °C for 2 min, as well as a final extension step at 72 °C for 10 min. After amplification, the PCR product mixture was digested by Bam HI as well as Hin dIII, and directly ligated into the pETBlue-2 (Novagen) expression vector cleaved with the same enzymes. .. The resulting recombinant plasmids were transferred into NovaBlue (Novagen) competent cells and placed on LB selection plates.

Article Title: Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms
Article Snippet: The PCR cycle consisted of an initial denaturation step of 96°C for 2 min, then 30 cycles of 94°C for 40 s, 57°C for 30 s, and 72°C for 2 min, followed by a final extension step at 72°C for 10 min. After amplification, the PCR product mixture was digested with Bam HI and Hin dIII, and ligated directly into pETBlue-2 (Novagen) expression vector cleaved with the same enzymes. .. The resultant recombinant plasmids were transferred into NovaBlue (Novagen) competent cells and plated onto LB selection plates.

Construct:

Article Title: Concatenated Metallothionein as a Clonable Gold Label for Electron Microscopy
Article Snippet: Since NcoI and AflIII produced equivalent DNA ends, the second copy of MT was inserted with removal of both restriction sites at the ligation junction. .. The products of the ligation reactions for both constructs were transformed into Novablue (Novagen) E. coli cells. .. Properly fused constructs were isolated by screening for over-expressed proteins of the expected molecular weight.

Article Title: Characterization of a stalled complex on the β-barrel assembly machine
Article Snippet: To construct pET9a BamD-His , a cassette containing the coding sequence of the BamD-His was inserted into pET9a. .. NovaBlue (Novagen) cells were transformed with 1 μL of the ligation product and plated onto LB plates containing 50 μg/mL kanamycin.

Article Title: Characterization of a stalled complex on the β-barrel assembly machine
Article Snippet: To construct pET9a FLAG 3 -BamA , a cassette containing the coding sequence of the FLAG3 tag was inserted into pET9a His-BamA to replace the original His6 tag. .. NovaBlue (Novagen) cells were transformed with 1 μL of digested PCR product and plated onto LB plates containing 50 μg/mL kanamycin.

Incubation:

Article Title: Engineered Cpf1 variants with altered PAM specificities increase genome targeting range
Article Snippet: NovaBlue(DE3) E. coli (Novagen) were transformed with the variant library and plated on LB agar containing 25 μg/mL chloramphenicol. .. Surviving colonies were scraped and cultured in ZymoBroth with 25 μg/mL chloramphenicol to an O.D. of 0.4–0.6 and made competent using a Mix & Go kit (Zymo).

Article Title: Association of Mycobacterium Proteins with Lipid Droplets
Article Snippet: Escherichia coli strains DH5α, Top10, (Thermo Fisher Scientific, Waltham, MA), and NovaBlue (MilliporeSigma, Burlington, MA) were used for cloning. .. Protein purification was carried out in E. coil strain BL21(DE3) containing plasmid pLysS.

Article Title: Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms
Article Snippet: The resultant recombinant plasmids were transferred into NovaBlue (Novagen) competent cells and plated onto LB selection plates. .. The transformed bacterial cells were cultured in LB medium containing 50 μg/ml carbenicillin and 100 μg/ml chloramphenicol at 37°C, and protein expression was induced by the addition of 1 mM isopropyl-β-D-thiogalactopyranoside when the optical density at 660 nm reached 0.6.

Activity Assay:

Article Title: Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms
Article Snippet: The fumF nucleotide sequence was amplified from the plasmid (pGXAM3566) isolated from a clone with fumarase activity. .. The resultant recombinant plasmids were transferred into NovaBlue (Novagen) competent cells and plated onto LB selection plates.

Expressing:

Article Title: Concatenated Metallothionein as a Clonable Gold Label for Electron Microscopy
Article Snippet: The products of the ligation reactions for both constructs were transformed into Novablue (Novagen) E. coli cells. .. Properly fused constructs were isolated by screening for over-expressed proteins of the expected molecular weight.

Article Title: Probing the Substrate Specificity and Protein-Protein Interactions of the E. coli Fatty Acid Dehydratase, FabA
Article Snippet: The FabA gene was cloned into the pET28b expression vector and used as the DNA template for site-directed mutagenesis with the mutagenic primer listed in . .. Digested PCR products were transformed into NovaBlue (EMD Millipore).

Article Title: An ATP-Grasp Ligase Involved in the Last Biosynthetic Step of the Iminomycosporine Shinorine in Nostoc punctiforme ATCC 29133
Article Snippet: Escherichia coli DH5α or NovaBlue (Novagen/EMD Biosciences, San Diego, CA) competent cells were used for standard subcloning. .. All E. coli strains were grown and transformed as described previously ( ).

Article Title: Characterization of a Novel Serine Protease Inhibitor Gene from a Marine Metagenome
Article Snippet: The PCR cycling consisted of denaturation steps (96 °C for 2 min), 30 cycles at 94 °C for 40 s, 58.5 °C for 30 s, and 72 °C for 2 min, as well as a final extension step at 72 °C for 10 min. After amplification, the PCR product mixture was digested by Bam HI as well as Hin dIII, and directly ligated into the pETBlue-2 (Novagen) expression vector cleaved with the same enzymes. .. The resulting recombinant plasmids were transferred into NovaBlue (Novagen) competent cells and placed on LB selection plates.

Article Title: Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms
Article Snippet: Paragraph title: Expression and purification of the recombinant fumarase protein ... The resultant recombinant plasmids were transferred into NovaBlue (Novagen) competent cells and plated onto LB selection plates.

Modification:

Article Title: Concatenated Metallothionein as a Clonable Gold Label for Electron Microscopy
Article Snippet: In this construct, the only translational modification was the change of an alanine to an aspartic acid at the junction between the two MT genes. .. The products of the ligation reactions for both constructs were transformed into Novablue (Novagen) E. coli cells.

Article Title: The communication between the zinc and nickel sites in dimeric HypA: Metal recognition and pH sensing
Article Snippet: The PCR product was used to transform into NovaBlue (Novagen™) competent cells. .. This DNA was then used to transform DL41 (DE3) pLysS (Novagen™) competent cells for protein overexpression.

Transformation Assay:

Article Title: Engineered Cpf1 variants with altered PAM specificities increase genome targeting range
Article Snippet: The variant library was assembled by cassette mutagenesis, mini-prepped, pooled, and precipitated with isopropanol. .. NovaBlue(DE3) E. coli (Novagen) were transformed with the variant library and plated on LB agar containing 25 μg/mL chloramphenicol. .. Surviving colonies were scraped and cultured in ZymoBroth with 25 μg/mL chloramphenicol to an O.D. of 0.4–0.6 and made competent using a Mix & Go kit (Zymo).

Article Title: Design of pH Sensitive Binding Proteins from the Hyperthermophilic Sso7d Scaffold
Article Snippet: Plasmid DNA was isolated from the Sso7d mutants using Zymoprep Kit II. .. The isolated DNA was further transformed into Novablue™ (E. coli ) cells (EMD Biosciences, San Diego, CA). .. A Qiagen miniprep kit was used to isolate plasmids from E.coli (Qiagen, Valencia, CA) and sequenced using the primer 5′ ACT ACG CTC TGC AGG CTA GT 3′.

Article Title: Concatenated Metallothionein as a Clonable Gold Label for Electron Microscopy
Article Snippet: Since NcoI and AflIII produced equivalent DNA ends, the second copy of MT was inserted with removal of both restriction sites at the ligation junction. .. The products of the ligation reactions for both constructs were transformed into Novablue (Novagen) E. coli cells. .. Properly fused constructs were isolated by screening for over-expressed proteins of the expected molecular weight.

Article Title:
Article Snippet: The cells were lysed, and plasmid DNA was extracted by using the Zymoprep yeast plasmid DNA miniprep II (Zymo Research). .. To improve the quality of the DNA, it was transformed into NovaBlue (Merck Novagen) bacteria, plated on LB agar with 100 mg liter−1 ampicillin. .. DNA was isolated from the bacteria using the QIAprep miniprep kit, and the DNA was sequenced by StarSEQ GmbH (Mainz, Germany) using the primer Seq-pCON-F.

Article Title: Characterization of a stalled complex on the β-barrel assembly machine
Article Snippet: The digested product was gel-purified and the coding sequence of the BamD-His was ligated to a pET9a vector that had been digested with XbaI and BlpI for > 3 h at 37 °C. .. NovaBlue (Novagen) cells were transformed with 1 μL of the ligation product and plated onto LB plates containing 50 μg/mL kanamycin. .. Proper plasmid construction was confirmed by DNA sequencing.

Article Title: Characterization of a stalled complex on the β-barrel assembly machine
Article Snippet: Briefly, the entire pET9a His-BamA template was amplified by PCR (using primers 5′-A GAT CAT GAT ATC GAC TAT AAA GAC GAT GAT GAC AAA GAA GGG TTC GTA GTG AAA GAT-3′ and 5′-TCG ATA TCA TGA TCT TTG TAG TCG CCG TCG TGA TCT TTA TAA TCA GCA CCG TAT ACG GTG-3′) and the resulting PCR product mixture digested with DpnI for > 1 h at 37 °C. .. NovaBlue (Novagen) cells were transformed with 1 μL of digested PCR product and plated onto LB plates containing 50 μg/mL kanamycin. .. Proper plasmid construction was confirmed by DNA sequencing.

Article Title: Probing the Substrate Specificity and Protein-Protein Interactions of the E. coli Fatty Acid Dehydratase, FabA
Article Snippet: FabA PCR products were digested with Dpn1 to eliminate template WT FabA. .. Digested PCR products were transformed into NovaBlue (EMD Millipore). .. Single colonies were isolated and their plasmids were extracted using Qiagen Minipreps.

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of His1 and its H-bonding Network
Article Snippet: The PCR product was gel purified (1% agarose) and treated with T4 DNA polymerase to create overhangs complementary to the Ligation Independent Cloning (LIC) vector pET30 Xa/LIC (Novagen™). .. The insert was annealed to the vector and NovaBlue (Novagen™) competent cells were transformed with the pET30 Xa/LIC plasmid containing the sodN mutant gene for selection. .. The plasmid containing mutagenic sodN was isolated using a plasmid mini prep kit (Qiagen) and DNA sequencing was performed to confirm the expected base sequence for the mutant (University of Massachusetts-Amherst DNA sequencing facility).

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of His1 and its H-bonding Network
Article Snippet: The R47A-NiSOD mutant protein was prepared by PCR of WT-NiSOD pET-30 Xa/LIC plasmid with mutagenic 5’- and 3’-primers and the E17A/R47A-NiSOD and E17R/R47E-NiSOD double mutations were generated by PCR of the R47A-NiSOD pET-30 Xa/LIC plasmid with mutagenic 5’- and 3’-primers using the QuikChange® XL Site-Directed Mutagenesis Kit (supporting information ). .. NovaBlue (Novagen™) competent cells were transformed with the PCR product for mutant gene for selection. .. The plasmid containing mutagenic sodN was isolated using a plasmid mini prep kit (Qiagen) and DNA sequencing was performed to confirm the expected base sequence for the mutant (W. M. Keck DNA facility at Yale University, New Haven, CT).

Article Title: An ATP-Grasp Ligase Involved in the Last Biosynthetic Step of the Iminomycosporine Shinorine in Nostoc punctiforme ATCC 29133
Article Snippet: Escherichia coli DH5α or NovaBlue (Novagen/EMD Biosciences, San Diego, CA) competent cells were used for standard subcloning. .. E. coli BL21(DE3) (Novagen) was used for gene expression and for in vivo bioconversion.

Article Title: Characterization of a Novel Serine Protease Inhibitor Gene from a Marine Metagenome
Article Snippet: The resulting recombinant plasmids were transferred into NovaBlue (Novagen) competent cells and placed on LB selection plates. .. The recombinant expression plasmid was then introduced into E. coli BL21(DE3)pLysS to express the target protein.

Article Title: Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms
Article Snippet: The resultant recombinant plasmids were transferred into NovaBlue (Novagen) competent cells and plated onto LB selection plates. .. After overnight incubation at 37°C, positive white colonies were picked for isolation of the recombinant expression plasmid, which was subsequently introduced into E. coli BL21(DE3)pLysS (Novagen) to express the target protein.

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of Cys2 and Cys6
Article Snippet: The PCR product was gel purified (1% agarose) and treated with T4 DNA polymerase to create overhangs complementary to the Ligation Independent Cloning (LIC) vector pET30 Xa/LIC (Novagen™ ). .. The insert was annealed to the vector and NovaBlue (Novagen™ ) competent cells were transformed with the pET30 Xa/LIC plasmid containing the sodN mutant gene for selection. .. The plasmid containing mutagenic sodN was isolated using plasmid mini prep kit from Qiagen and DNA sequencing was performed to confirm the expected base sequence for each mutant (University of Massachusetts-Amherst DNA sequencing facility).

Over Expression:

Article Title: The communication between the zinc and nickel sites in dimeric HypA: Metal recognition and pH sensing
Article Snippet: The PCR product was used to transform into NovaBlue (Novagen™) competent cells. .. The PCR product was used to transform into NovaBlue (Novagen™) competent cells.

Article Title: Characterization of a Novel Serine Protease Inhibitor Gene from a Marine Metagenome
Article Snippet: Paragraph title: 3.4. Overexpression and Purification of the Recombinant Serpin Protein ... The resulting recombinant plasmids were transferred into NovaBlue (Novagen) competent cells and placed on LB selection plates.

Ligation:

Article Title: Concatenated Metallothionein as a Clonable Gold Label for Electron Microscopy
Article Snippet: Since NcoI and AflIII produced equivalent DNA ends, the second copy of MT was inserted with removal of both restriction sites at the ligation junction. .. The products of the ligation reactions for both constructs were transformed into Novablue (Novagen) E. coli cells. .. Properly fused constructs were isolated by screening for over-expressed proteins of the expected molecular weight.

Article Title: Characterization of a stalled complex on the β-barrel assembly machine
Article Snippet: The digested product was gel-purified and the coding sequence of the BamD-His was ligated to a pET9a vector that had been digested with XbaI and BlpI for > 3 h at 37 °C. .. NovaBlue (Novagen) cells were transformed with 1 μL of the ligation product and plated onto LB plates containing 50 μg/mL kanamycin. .. Proper plasmid construction was confirmed by DNA sequencing.

Article Title: Characterization of a stalled complex on the β-barrel assembly machine
Article Snippet: NovaBlue (Novagen) cells were transformed with 1 μL of the ligation product and plated onto LB plates containing 50 μg/mL kanamycin. .. NovaBlue (Novagen) cells were transformed with 1 μL of digested PCR product and plated onto LB plates containing 50 μg/mL kanamycin.

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of His1 and its H-bonding Network
Article Snippet: The PCR product was gel purified (1% agarose) and treated with T4 DNA polymerase to create overhangs complementary to the Ligation Independent Cloning (LIC) vector pET30 Xa/LIC (Novagen™). .. The insert was annealed to the vector and NovaBlue (Novagen™) competent cells were transformed with the pET30 Xa/LIC plasmid containing the sodN mutant gene for selection.

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of Cys2 and Cys6
Article Snippet: The PCR product was gel purified (1% agarose) and treated with T4 DNA polymerase to create overhangs complementary to the Ligation Independent Cloning (LIC) vector pET30 Xa/LIC (Novagen™ ). .. The insert was annealed to the vector and NovaBlue (Novagen™ ) competent cells were transformed with the pET30 Xa/LIC plasmid containing the sodN mutant gene for selection.

Cell Culture:

Article Title:
Article Snippet: Selected cells were centrifuged at 3000 × g for 5 min to remove the sheath fluid and then cultured at 30 °C for 1 or 2 days in SD-CAA, after which DNA was extracted for deep sequencing (see below). .. To improve the quality of the DNA, it was transformed into NovaBlue (Merck Novagen) bacteria, plated on LB agar with 100 mg liter−1 ampicillin.

Article Title: Enhancement of astaxanthin production in Xanthophyllomyces dendrorhous by efficient method for the complete deletion of genes
Article Snippet: NovaBlue (Novagen, Madison, WI, USA) was used as the E. coli host strain for recombinant DNA manipulation. .. NovaBlue (Novagen, Madison, WI, USA) was used as the E. coli host strain for recombinant DNA manipulation.

Article Title: Association of Mycobacterium Proteins with Lipid Droplets
Article Snippet: Escherichia coli strains DH5α, Top10, (Thermo Fisher Scientific, Waltham, MA), and NovaBlue (MilliporeSigma, Burlington, MA) were used for cloning. .. All E. coli strains were incubated at 37°C in Luria-Bertani (LB) medium with 100 μg/ml ampicillin, 50 μg/ml kanamycin, 150 μg/ml hygromycin B, and/or 25 μg/ml chloramphenicol for selection, as necessary.

Article Title: Development of a multi-gene expression system in Xanthophyllomyces dendrorhous
Article Snippet: NovaBlue (Novagen, Madison, WI, USA) was used as the Escherichia coli host strain for recombinant DNA manipulation. .. NovaBlue (Novagen, Madison, WI, USA) was used as the Escherichia coli host strain for recombinant DNA manipulation.

Article Title: Characterization of a Novel Serine Protease Inhibitor Gene from a Marine Metagenome
Article Snippet: The resulting recombinant plasmids were transferred into NovaBlue (Novagen) competent cells and placed on LB selection plates. .. The recombinant expression plasmid was then introduced into E. coli BL21(DE3)pLysS to express the target protein.

Article Title: Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms
Article Snippet: The resultant recombinant plasmids were transferred into NovaBlue (Novagen) competent cells and plated onto LB selection plates. .. After overnight incubation at 37°C, positive white colonies were picked for isolation of the recombinant expression plasmid, which was subsequently introduced into E. coli BL21(DE3)pLysS (Novagen) to express the target protein.

Generated:

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of His1 and its H-bonding Network
Article Snippet: The insert was annealed to the vector and NovaBlue (Novagen™) competent cells were transformed with the pET30 Xa/LIC plasmid containing the sodN mutant gene for selection. .. The plasmid containing mutagenic sodN was isolated using a plasmid mini prep kit (Qiagen) and DNA sequencing was performed to confirm the expected base sequence for the mutant (University of Massachusetts-Amherst DNA sequencing facility).

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of His1 and its H-bonding Network
Article Snippet: The R47A-NiSOD mutant protein was prepared by PCR of WT-NiSOD pET-30 Xa/LIC plasmid with mutagenic 5’- and 3’-primers and the E17A/R47A-NiSOD and E17R/R47E-NiSOD double mutations were generated by PCR of the R47A-NiSOD pET-30 Xa/LIC plasmid with mutagenic 5’- and 3’-primers using the QuikChange® XL Site-Directed Mutagenesis Kit (supporting information ). .. NovaBlue (Novagen™) competent cells were transformed with the PCR product for mutant gene for selection.

DNA Sequencing:

Article Title: Characterization of a stalled complex on the β-barrel assembly machine
Article Snippet: NovaBlue (Novagen) cells were transformed with 1 μL of the ligation product and plated onto LB plates containing 50 μg/mL kanamycin. .. NovaBlue (Novagen) cells were transformed with 1 μL of the ligation product and plated onto LB plates containing 50 μg/mL kanamycin.

Article Title: Characterization of a stalled complex on the β-barrel assembly machine
Article Snippet: NovaBlue (Novagen) cells were transformed with 1 μL of digested PCR product and plated onto LB plates containing 50 μg/mL kanamycin. .. NovaBlue (Novagen) cells were transformed with 1 μL of digested PCR product and plated onto LB plates containing 50 μg/mL kanamycin.

Article Title: Probing the Substrate Specificity and Protein-Protein Interactions of the E. coli Fatty Acid Dehydratase, FabA
Article Snippet: Digested PCR products were transformed into NovaBlue (EMD Millipore). .. Single colonies were isolated and their plasmids were extracted using Qiagen Minipreps.

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of His1 and its H-bonding Network
Article Snippet: The insert was annealed to the vector and NovaBlue (Novagen™) competent cells were transformed with the pET30 Xa/LIC plasmid containing the sodN mutant gene for selection. .. The insert was annealed to the vector and NovaBlue (Novagen™) competent cells were transformed with the pET30 Xa/LIC plasmid containing the sodN mutant gene for selection.

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of His1 and its H-bonding Network
Article Snippet: The plasmid containing mutagenic sodN was isolated using a plasmid mini prep kit (Qiagen) and DNA sequencing was performed to confirm the expected base sequence for the mutant (University of Massachusetts-Amherst DNA sequencing facility). .. NovaBlue (Novagen™) competent cells were transformed with the PCR product for mutant gene for selection.

Polymerase Chain Reaction:

Article Title: Concatenated Metallothionein as a Clonable Gold Label for Electron Microscopy
Article Snippet: The pMAL-c2x-MT plasmid was prepared by digesting with XmnI and NcoI, and the PCR product was constructed with XmnI and AflIII. .. The products of the ligation reactions for both constructs were transformed into Novablue (Novagen) E. coli cells.

Article Title: Characterization of a stalled complex on the β-barrel assembly machine
Article Snippet: NovaBlue (Novagen) cells were transformed with 1 μL of the ligation product and plated onto LB plates containing 50 μg/mL kanamycin. .. To construct pET9a FLAG 3 -BamA , a cassette containing the coding sequence of the FLAG3 tag was inserted into pET9a His-BamA to replace the original His6 tag.

Article Title: Characterization of a stalled complex on the β-barrel assembly machine
Article Snippet: Briefly, the entire pET9a His-BamA template was amplified by PCR (using primers 5′-A GAT CAT GAT ATC GAC TAT AAA GAC GAT GAT GAC AAA GAA GGG TTC GTA GTG AAA GAT-3′ and 5′-TCG ATA TCA TGA TCT TTG TAG TCG CCG TCG TGA TCT TTA TAA TCA GCA CCG TAT ACG GTG-3′) and the resulting PCR product mixture digested with DpnI for > 1 h at 37 °C. .. NovaBlue (Novagen) cells were transformed with 1 μL of digested PCR product and plated onto LB plates containing 50 μg/mL kanamycin. .. Proper plasmid construction was confirmed by DNA sequencing.

Article Title: Probing the Substrate Specificity and Protein-Protein Interactions of the E. coli Fatty Acid Dehydratase, FabA
Article Snippet: FabA PCR products were digested with Dpn1 to eliminate template WT FabA. .. Digested PCR products were transformed into NovaBlue (EMD Millipore). .. Single colonies were isolated and their plasmids were extracted using Qiagen Minipreps.

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of His1 and its H-bonding Network
Article Snippet: The PCR product was gel purified (1% agarose) and treated with T4 DNA polymerase to create overhangs complementary to the Ligation Independent Cloning (LIC) vector pET30 Xa/LIC (Novagen™). .. The insert was annealed to the vector and NovaBlue (Novagen™) competent cells were transformed with the pET30 Xa/LIC plasmid containing the sodN mutant gene for selection.

Article Title: The communication between the zinc and nickel sites in dimeric HypA: Metal recognition and pH sensing
Article Snippet: Mutagenic primers and the parental DNA that were used in the PCR reactions are tabulated in the . .. The PCR product was used to transform into NovaBlue (Novagen™) competent cells. .. After transformation, the cells were grown on LB/amp agar plates overnight at 37° C. Three separate colonies were selected from the plate and grown in a 5 mL culture (LB amp media) overnight.

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of His1 and its H-bonding Network
Article Snippet: The R47A-NiSOD mutant protein was prepared by PCR of WT-NiSOD pET-30 Xa/LIC plasmid with mutagenic 5’- and 3’-primers and the E17A/R47A-NiSOD and E17R/R47E-NiSOD double mutations were generated by PCR of the R47A-NiSOD pET-30 Xa/LIC plasmid with mutagenic 5’- and 3’-primers using the QuikChange® XL Site-Directed Mutagenesis Kit (supporting information ). .. NovaBlue (Novagen™) competent cells were transformed with the PCR product for mutant gene for selection. .. The plasmid containing mutagenic sodN was isolated using a plasmid mini prep kit (Qiagen) and DNA sequencing was performed to confirm the expected base sequence for the mutant (W. M. Keck DNA facility at Yale University, New Haven, CT).

Article Title: Characterization of a Novel Serine Protease Inhibitor Gene from a Marine Metagenome
Article Snippet: The PCR cycling consisted of denaturation steps (96 °C for 2 min), 30 cycles at 94 °C for 40 s, 58.5 °C for 30 s, and 72 °C for 2 min, as well as a final extension step at 72 °C for 10 min. After amplification, the PCR product mixture was digested by Bam HI as well as Hin dIII, and directly ligated into the pETBlue-2 (Novagen) expression vector cleaved with the same enzymes. .. The resulting recombinant plasmids were transferred into NovaBlue (Novagen) competent cells and placed on LB selection plates.

Article Title: Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms
Article Snippet: The PCR cycle consisted of an initial denaturation step of 96°C for 2 min, then 30 cycles of 94°C for 40 s, 57°C for 30 s, and 72°C for 2 min, followed by a final extension step at 72°C for 10 min. After amplification, the PCR product mixture was digested with Bam HI and Hin dIII, and ligated directly into pETBlue-2 (Novagen) expression vector cleaved with the same enzymes. .. The resultant recombinant plasmids were transferred into NovaBlue (Novagen) competent cells and plated onto LB selection plates.

Article Title: The Effects of Select Histidine to Cysteine Mutations on Transcriptional Regulation by E. coli RcnR
Article Snippet: Primers used in the PCR reactions are shown in the . .. The PCR product was transformed into NovaBlue (Novagen) competent cells. .. The cells were then grown on LB/agar plates overnight, a single colony was selected and grown overnight in a 5 mL culture (LB amp media).

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of Cys2 and Cys6
Article Snippet: The PCR product was gel purified (1% agarose) and treated with T4 DNA polymerase to create overhangs complementary to the Ligation Independent Cloning (LIC) vector pET30 Xa/LIC (Novagen™ ). .. The insert was annealed to the vector and NovaBlue (Novagen™ ) competent cells were transformed with the pET30 Xa/LIC plasmid containing the sodN mutant gene for selection.

Sonication:

Article Title: Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms
Article Snippet: The resultant recombinant plasmids were transferred into NovaBlue (Novagen) competent cells and plated onto LB selection plates. .. The transformed bacterial cells were cultured in LB medium containing 50 μg/ml carbenicillin and 100 μg/ml chloramphenicol at 37°C, and protein expression was induced by the addition of 1 mM isopropyl-β-D-thiogalactopyranoside when the optical density at 660 nm reached 0.6.

Binding Assay:

Article Title: Design of pH Sensitive Binding Proteins from the Hyperthermophilic Sso7d Scaffold
Article Snippet: Mutants that lost binding to hFc-biotin were selected. .. The isolated DNA was further transformed into Novablue™ (E. coli ) cells (EMD Biosciences, San Diego, CA).

Article Title: The communication between the zinc and nickel sites in dimeric HypA: Metal recognition and pH sensing
Article Snippet: Point mutations of Cys residues in the two CxxC motifs associated with zinc binding and the two His residues (His79 and His95) that flank these two CxxC motifs were accomplished using the QuikChange (Stratagene™) mutagenesis kit and the pJI100 plasmid. .. The PCR product was used to transform into NovaBlue (Novagen™) competent cells.

Molecular Weight:

Article Title: Concatenated Metallothionein as a Clonable Gold Label for Electron Microscopy
Article Snippet: The products of the ligation reactions for both constructs were transformed into Novablue (Novagen) E. coli cells. .. Isolated colonies grown in LB (5 gr yeast extract, 10 gr tryptone, and 5 gr sodium chloride in 1 L) with selection were checked for expression after induction with 1mM isopropyl-B-D-thiogalactopyranoside (IPTG) for 2hr.

Mutagenesis:

Article Title: Engineered Cpf1 variants with altered PAM specificities increase genome targeting range
Article Snippet: NovaBlue(DE3) E. coli (Novagen) were transformed with the variant library and plated on LB agar containing 25 μg/mL chloramphenicol. .. Surviving colonies were scraped and cultured in ZymoBroth with 25 μg/mL chloramphenicol to an O.D. of 0.4–0.6 and made competent using a Mix & Go kit (Zymo).

Article Title: Design of pH Sensitive Binding Proteins from the Hyperthermophilic Sso7d Scaffold
Article Snippet: Paragraph title: Random Mutagenesis Library Construction and Cell Sorting ... The isolated DNA was further transformed into Novablue™ (E. coli ) cells (EMD Biosciences, San Diego, CA).

Article Title: Probing the Substrate Specificity and Protein-Protein Interactions of the E. coli Fatty Acid Dehydratase, FabA
Article Snippet: Paragraph title: Site-directed Mutagenesis ... Digested PCR products were transformed into NovaBlue (EMD Millipore).

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of His1 and its H-bonding Network
Article Snippet: The PCR product was gel purified (1% agarose) and treated with T4 DNA polymerase to create overhangs complementary to the Ligation Independent Cloning (LIC) vector pET30 Xa/LIC (Novagen™). .. The insert was annealed to the vector and NovaBlue (Novagen™) competent cells were transformed with the pET30 Xa/LIC plasmid containing the sodN mutant gene for selection. .. The plasmid containing mutagenic sodN was isolated using a plasmid mini prep kit (Qiagen) and DNA sequencing was performed to confirm the expected base sequence for the mutant (University of Massachusetts-Amherst DNA sequencing facility).

Article Title: The communication between the zinc and nickel sites in dimeric HypA: Metal recognition and pH sensing
Article Snippet: Paragraph title: HypA mutagenesis and protein characterization ... The PCR product was used to transform into NovaBlue (Novagen™) competent cells.

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of His1 and its H-bonding Network
Article Snippet: The R47A-NiSOD mutant protein was prepared by PCR of WT-NiSOD pET-30 Xa/LIC plasmid with mutagenic 5’- and 3’-primers and the E17A/R47A-NiSOD and E17R/R47E-NiSOD double mutations were generated by PCR of the R47A-NiSOD pET-30 Xa/LIC plasmid with mutagenic 5’- and 3’-primers using the QuikChange® XL Site-Directed Mutagenesis Kit (supporting information ). .. NovaBlue (Novagen™) competent cells were transformed with the PCR product for mutant gene for selection. .. The plasmid containing mutagenic sodN was isolated using a plasmid mini prep kit (Qiagen) and DNA sequencing was performed to confirm the expected base sequence for the mutant (W. M. Keck DNA facility at Yale University, New Haven, CT).

Article Title: The Effects of Select Histidine to Cysteine Mutations on Transcriptional Regulation by E. coli RcnR
Article Snippet: Paragraph title: RcnR Mutagenesis ... The PCR product was transformed into NovaBlue (Novagen) competent cells.

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of Cys2 and Cys6
Article Snippet: The PCR product was gel purified (1% agarose) and treated with T4 DNA polymerase to create overhangs complementary to the Ligation Independent Cloning (LIC) vector pET30 Xa/LIC (Novagen™ ). .. The insert was annealed to the vector and NovaBlue (Novagen™ ) competent cells were transformed with the pET30 Xa/LIC plasmid containing the sodN mutant gene for selection. .. The plasmid containing mutagenic sodN was isolated using plasmid mini prep kit from Qiagen and DNA sequencing was performed to confirm the expected base sequence for each mutant (University of Massachusetts-Amherst DNA sequencing facility).

Isolation:

Article Title: Engineered Cpf1 variants with altered PAM specificities increase genome targeting range
Article Snippet: NovaBlue(DE3) E. coli (Novagen) were transformed with the variant library and plated on LB agar containing 25 μg/mL chloramphenicol. .. A negative control was obtained by transforming the E. coli with pUC19, which lacks the target site.

Article Title: Design of pH Sensitive Binding Proteins from the Hyperthermophilic Sso7d Scaffold
Article Snippet: Plasmid DNA was isolated from the Sso7d mutants using Zymoprep Kit II. .. The isolated DNA was further transformed into Novablue™ (E. coli ) cells (EMD Biosciences, San Diego, CA). .. A Qiagen miniprep kit was used to isolate plasmids from E.coli (Qiagen, Valencia, CA) and sequenced using the primer 5′ ACT ACG CTC TGC AGG CTA GT 3′.

Article Title: Concatenated Metallothionein as a Clonable Gold Label for Electron Microscopy
Article Snippet: The products of the ligation reactions for both constructs were transformed into Novablue (Novagen) E. coli cells. .. Properly fused constructs were isolated by screening for over-expressed proteins of the expected molecular weight.

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of His1 and its H-bonding Network
Article Snippet: The insert was annealed to the vector and NovaBlue (Novagen™) competent cells were transformed with the pET30 Xa/LIC plasmid containing the sodN mutant gene for selection. .. The insert was annealed to the vector and NovaBlue (Novagen™) competent cells were transformed with the pET30 Xa/LIC plasmid containing the sodN mutant gene for selection.

Article Title: The communication between the zinc and nickel sites in dimeric HypA: Metal recognition and pH sensing
Article Snippet: The PCR product was used to transform into NovaBlue (Novagen™) competent cells. .. After transformation, the cells were grown on LB/amp agar plates overnight at 37° C. Three separate colonies were selected from the plate and grown in a 5 mL culture (LB amp media) overnight.

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of His1 and its H-bonding Network
Article Snippet: The plasmid containing mutagenic sodN was isolated using a plasmid mini prep kit (Qiagen) and DNA sequencing was performed to confirm the expected base sequence for the mutant (University of Massachusetts-Amherst DNA sequencing facility). .. NovaBlue (Novagen™) competent cells were transformed with the PCR product for mutant gene for selection.

Article Title: Characterization of a Novel Serine Protease Inhibitor Gene from a Marine Metagenome
Article Snippet: The Spi1C nucleotide sequence was amplified from the plasmid pGXAG59 isolated from a serpin-producing clone. .. The resulting recombinant plasmids were transferred into NovaBlue (Novagen) competent cells and placed on LB selection plates.

Article Title: Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms
Article Snippet: The fumF nucleotide sequence was amplified from the plasmid (pGXAM3566) isolated from a clone with fumarase activity. .. The resultant recombinant plasmids were transferred into NovaBlue (Novagen) competent cells and plated onto LB selection plates.

Subcloning:

Article Title: An ATP-Grasp Ligase Involved in the Last Biosynthetic Step of the Iminomycosporine Shinorine in Nostoc punctiforme ATCC 29133
Article Snippet: UV-B radiation was provided by 40-W fluorescent tubes (UV-B-313EL; Q-Lab Corporation, Cleveland, OH) at an approximate intensity of 0.9 W/m−2 . .. Escherichia coli DH5α or NovaBlue (Novagen/EMD Biosciences, San Diego, CA) competent cells were used for standard subcloning. .. E. coli BL21(DE3) (Novagen) was used for gene expression and for in vivo bioconversion.

Labeling:

Article Title: Design of pH Sensitive Binding Proteins from the Hyperthermophilic Sso7d Scaffold
Article Snippet: The sorted population was labeled with 100 nM hFc-biotin in P2-BSA and cells that lost binding to hFc-biotin were selected. .. The isolated DNA was further transformed into Novablue™ (E. coli ) cells (EMD Biosciences, San Diego, CA).

Purification:

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of His1 and its H-bonding Network
Article Snippet: The PCR product was gel purified (1% agarose) and treated with T4 DNA polymerase to create overhangs complementary to the Ligation Independent Cloning (LIC) vector pET30 Xa/LIC (Novagen™). .. The insert was annealed to the vector and NovaBlue (Novagen™) competent cells were transformed with the pET30 Xa/LIC plasmid containing the sodN mutant gene for selection.

Article Title: The communication between the zinc and nickel sites in dimeric HypA: Metal recognition and pH sensing
Article Snippet: The PCR product was used to transform into NovaBlue (Novagen™) competent cells. .. The PCR product was used to transform into NovaBlue (Novagen™) competent cells.

Article Title: Mutations in RNA Polymerase Bridge Helix and Switch Regions Affect Active-Site Networks and Transcript-Assisted Hydrolysis
Article Snippet: E . coli rpoB and rpoC genes harbouring single substitutions were mutagenised in plasmids pIA458, pIA545 and pIA661 and were sub-cloned into pVS10 vectors . .. The reconstituted RNAP mutants were over-expressed in NovaBlue (Novagen) and purified as previously described . .. E . coli PspF1 -275 (the AAA+ domain, residues 1–275) and Klebsiella pneumoniae σ54 were over-expressed in BL21(DE3) and purified as previously described .

Article Title: Characterization of a Novel Serine Protease Inhibitor Gene from a Marine Metagenome
Article Snippet: Paragraph title: 3.4. Overexpression and Purification of the Recombinant Serpin Protein ... The resulting recombinant plasmids were transferred into NovaBlue (Novagen) competent cells and placed on LB selection plates.

Article Title: Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms
Article Snippet: Paragraph title: Expression and purification of the recombinant fumarase protein ... The resultant recombinant plasmids were transferred into NovaBlue (Novagen) competent cells and plated onto LB selection plates.

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of Cys2 and Cys6
Article Snippet: The PCR product was gel purified (1% agarose) and treated with T4 DNA polymerase to create overhangs complementary to the Ligation Independent Cloning (LIC) vector pET30 Xa/LIC (Novagen™ ). .. The insert was annealed to the vector and NovaBlue (Novagen™ ) competent cells were transformed with the pET30 Xa/LIC plasmid containing the sodN mutant gene for selection.

Sequencing:

Article Title: Design of pH Sensitive Binding Proteins from the Hyperthermophilic Sso7d Scaffold
Article Snippet: Cells from the final sort were plated on SDCAA plates and individual clones were picked for sequencing. .. The isolated DNA was further transformed into Novablue™ (E. coli ) cells (EMD Biosciences, San Diego, CA).

Article Title:
Article Snippet: Selected cells were centrifuged at 3000 × g for 5 min to remove the sheath fluid and then cultured at 30 °C for 1 or 2 days in SD-CAA, after which DNA was extracted for deep sequencing (see below). .. To improve the quality of the DNA, it was transformed into NovaBlue (Merck Novagen) bacteria, plated on LB agar with 100 mg liter−1 ampicillin.

Article Title: Characterization of a stalled complex on the β-barrel assembly machine
Article Snippet: The digested product was gel-purified and the coding sequence of the BamD-His was ligated to a pET9a vector that had been digested with XbaI and BlpI for > 3 h at 37 °C. .. NovaBlue (Novagen) cells were transformed with 1 μL of the ligation product and plated onto LB plates containing 50 μg/mL kanamycin.

Article Title: Characterization of a stalled complex on the β-barrel assembly machine
Article Snippet: To construct pET9a FLAG 3 -BamA , a cassette containing the coding sequence of the FLAG3 tag was inserted into pET9a His-BamA to replace the original His6 tag. .. NovaBlue (Novagen) cells were transformed with 1 μL of digested PCR product and plated onto LB plates containing 50 μg/mL kanamycin.

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of His1 and its H-bonding Network
Article Snippet: The insert was annealed to the vector and NovaBlue (Novagen™) competent cells were transformed with the pET30 Xa/LIC plasmid containing the sodN mutant gene for selection. .. The insert was annealed to the vector and NovaBlue (Novagen™) competent cells were transformed with the pET30 Xa/LIC plasmid containing the sodN mutant gene for selection.

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of His1 and its H-bonding Network
Article Snippet: The plasmid containing mutagenic sodN was isolated using a plasmid mini prep kit (Qiagen) and DNA sequencing was performed to confirm the expected base sequence for the mutant (University of Massachusetts-Amherst DNA sequencing facility). .. NovaBlue (Novagen™) competent cells were transformed with the PCR product for mutant gene for selection.

Article Title: Characterization of a Novel Serine Protease Inhibitor Gene from a Marine Metagenome
Article Snippet: The Spi1C nucleotide sequence was amplified from the plasmid pGXAG59 isolated from a serpin-producing clone. .. The resulting recombinant plasmids were transferred into NovaBlue (Novagen) competent cells and placed on LB selection plates.

Article Title: Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms
Article Snippet: The fumF nucleotide sequence was amplified from the plasmid (pGXAM3566) isolated from a clone with fumarase activity. .. The resultant recombinant plasmids were transferred into NovaBlue (Novagen) competent cells and plated onto LB selection plates.

Selection:

Article Title: Engineered Cpf1 variants with altered PAM specificities increase genome targeting range
Article Snippet: Paragraph title: E. coli negative selection screen ... NovaBlue(DE3) E. coli (Novagen) were transformed with the variant library and plated on LB agar containing 25 μg/mL chloramphenicol.

Article Title: Concatenated Metallothionein as a Clonable Gold Label for Electron Microscopy
Article Snippet: The products of the ligation reactions for both constructs were transformed into Novablue (Novagen) E. coli cells. .. Properly fused constructs were isolated by screening for over-expressed proteins of the expected molecular weight.

Article Title:
Article Snippet: A third selection was done with the purity mode set to “single cell,” in which 100 cells were spotted directly on an SD-CAA agar plate, with only a single cell spotted per position on the plate. .. To improve the quality of the DNA, it was transformed into NovaBlue (Merck Novagen) bacteria, plated on LB agar with 100 mg liter−1 ampicillin.

Article Title: Association of Mycobacterium Proteins with Lipid Droplets
Article Snippet: Escherichia coli strains DH5α, Top10, (Thermo Fisher Scientific, Waltham, MA), and NovaBlue (MilliporeSigma, Burlington, MA) were used for cloning. .. Protein purification was carried out in E. coil strain BL21(DE3) containing plasmid pLysS.

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of His1 and its H-bonding Network
Article Snippet: The PCR product was gel purified (1% agarose) and treated with T4 DNA polymerase to create overhangs complementary to the Ligation Independent Cloning (LIC) vector pET30 Xa/LIC (Novagen™). .. The insert was annealed to the vector and NovaBlue (Novagen™) competent cells were transformed with the pET30 Xa/LIC plasmid containing the sodN mutant gene for selection. .. The plasmid containing mutagenic sodN was isolated using a plasmid mini prep kit (Qiagen) and DNA sequencing was performed to confirm the expected base sequence for the mutant (University of Massachusetts-Amherst DNA sequencing facility).

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of His1 and its H-bonding Network
Article Snippet: The R47A-NiSOD mutant protein was prepared by PCR of WT-NiSOD pET-30 Xa/LIC plasmid with mutagenic 5’- and 3’-primers and the E17A/R47A-NiSOD and E17R/R47E-NiSOD double mutations were generated by PCR of the R47A-NiSOD pET-30 Xa/LIC plasmid with mutagenic 5’- and 3’-primers using the QuikChange® XL Site-Directed Mutagenesis Kit (supporting information ). .. NovaBlue (Novagen™) competent cells were transformed with the PCR product for mutant gene for selection. .. The plasmid containing mutagenic sodN was isolated using a plasmid mini prep kit (Qiagen) and DNA sequencing was performed to confirm the expected base sequence for the mutant (W. M. Keck DNA facility at Yale University, New Haven, CT).

Article Title: Characterization of a Novel Serine Protease Inhibitor Gene from a Marine Metagenome
Article Snippet: The PCR cycling consisted of denaturation steps (96 °C for 2 min), 30 cycles at 94 °C for 40 s, 58.5 °C for 30 s, and 72 °C for 2 min, as well as a final extension step at 72 °C for 10 min. After amplification, the PCR product mixture was digested by Bam HI as well as Hin dIII, and directly ligated into the pETBlue-2 (Novagen) expression vector cleaved with the same enzymes. .. The resulting recombinant plasmids were transferred into NovaBlue (Novagen) competent cells and placed on LB selection plates. .. After overnight incubation at 37 °C, positive white colonies were picked for the isolation of the recombinant expression plasmid.

Article Title: Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms
Article Snippet: The PCR cycle consisted of an initial denaturation step of 96°C for 2 min, then 30 cycles of 94°C for 40 s, 57°C for 30 s, and 72°C for 2 min, followed by a final extension step at 72°C for 10 min. After amplification, the PCR product mixture was digested with Bam HI and Hin dIII, and ligated directly into pETBlue-2 (Novagen) expression vector cleaved with the same enzymes. .. The resultant recombinant plasmids were transferred into NovaBlue (Novagen) competent cells and plated onto LB selection plates. .. After overnight incubation at 37°C, positive white colonies were picked for isolation of the recombinant expression plasmid, which was subsequently introduced into E. coli BL21(DE3)pLysS (Novagen) to express the target protein.

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of Cys2 and Cys6
Article Snippet: The PCR product was gel purified (1% agarose) and treated with T4 DNA polymerase to create overhangs complementary to the Ligation Independent Cloning (LIC) vector pET30 Xa/LIC (Novagen™ ). .. The insert was annealed to the vector and NovaBlue (Novagen™ ) competent cells were transformed with the pET30 Xa/LIC plasmid containing the sodN mutant gene for selection. .. The plasmid containing mutagenic sodN was isolated using plasmid mini prep kit from Qiagen and DNA sequencing was performed to confirm the expected base sequence for each mutant (University of Massachusetts-Amherst DNA sequencing facility).

Affinity Column:

Article Title: Mutations in RNA Polymerase Bridge Helix and Switch Regions Affect Active-Site Networks and Transcript-Assisted Hydrolysis
Article Snippet: The reconstituted RNAP mutants were over-expressed in NovaBlue (Novagen) and purified as previously described . .. E . coli PspF1 -275 (the AAA+ domain, residues 1–275) and Klebsiella pneumoniae σ54 were over-expressed in BL21(DE3) and purified as previously described .

FACS:

Article Title: Design of pH Sensitive Binding Proteins from the Hyperthermophilic Sso7d Scaffold
Article Snippet: Paragraph title: Random Mutagenesis Library Construction and Cell Sorting ... The isolated DNA was further transformed into Novablue™ (E. coli ) cells (EMD Biosciences, San Diego, CA).

Article Title:
Article Snippet: Paragraph title: Yeast Display and FACS ... To improve the quality of the DNA, it was transformed into NovaBlue (Merck Novagen) bacteria, plated on LB agar with 100 mg liter−1 ampicillin.

Chloramphenicol Acetyltransferase Assay:

Article Title: Characterization of a stalled complex on the β-barrel assembly machine
Article Snippet: NovaBlue (Novagen) cells were transformed with 1 μL of the ligation product and plated onto LB plates containing 50 μg/mL kanamycin. .. To construct pET9a FLAG 3 -BamA , a cassette containing the coding sequence of the FLAG3 tag was inserted into pET9a His-BamA to replace the original His6 tag.

Article Title: Characterization of a stalled complex on the β-barrel assembly machine
Article Snippet: Briefly, the entire pET9a His-BamA template was amplified by PCR (using primers 5′-A GAT CAT GAT ATC GAC TAT AAA GAC GAT GAT GAC AAA GAA GGG TTC GTA GTG AAA GAT-3′ and 5′-TCG ATA TCA TGA TCT TTG TAG TCG CCG TCG TGA TCT TTA TAA TCA GCA CCG TAT ACG GTG-3′) and the resulting PCR product mixture digested with DpnI for > 1 h at 37 °C. .. NovaBlue (Novagen) cells were transformed with 1 μL of digested PCR product and plated onto LB plates containing 50 μg/mL kanamycin.

Plasmid Preparation:

Article Title: Engineered Cpf1 variants with altered PAM specificities increase genome targeting range
Article Snippet: NovaBlue(DE3) E. coli (Novagen) were transformed with the variant library and plated on LB agar containing 25 μg/mL chloramphenicol. .. Surviving colonies were scraped and cultured in ZymoBroth with 25 μg/mL chloramphenicol to an O.D. of 0.4–0.6 and made competent using a Mix & Go kit (Zymo).

Article Title: Design of pH Sensitive Binding Proteins from the Hyperthermophilic Sso7d Scaffold
Article Snippet: Plasmid DNA was isolated from the Sso7d mutants using Zymoprep Kit II. .. The isolated DNA was further transformed into Novablue™ (E. coli ) cells (EMD Biosciences, San Diego, CA).

Article Title: Concatenated Metallothionein as a Clonable Gold Label for Electron Microscopy
Article Snippet: The pMAL-c2x-MT plasmid was prepared by digesting with XmnI and NcoI, and the PCR product was constructed with XmnI and AflIII. .. The products of the ligation reactions for both constructs were transformed into Novablue (Novagen) E. coli cells.

Article Title:
Article Snippet: The cells were lysed, and plasmid DNA was extracted by using the Zymoprep yeast plasmid DNA miniprep II (Zymo Research). .. To improve the quality of the DNA, it was transformed into NovaBlue (Merck Novagen) bacteria, plated on LB agar with 100 mg liter−1 ampicillin.

Article Title: Characterization of a stalled complex on the β-barrel assembly machine
Article Snippet: Paragraph title: Plasmid Construction. ... NovaBlue (Novagen) cells were transformed with 1 μL of the ligation product and plated onto LB plates containing 50 μg/mL kanamycin.

Article Title: Probing the Substrate Specificity and Protein-Protein Interactions of the E. coli Fatty Acid Dehydratase, FabA
Article Snippet: The FabA gene was cloned into the pET28b expression vector and used as the DNA template for site-directed mutagenesis with the mutagenic primer listed in . .. Digested PCR products were transformed into NovaBlue (EMD Millipore).

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of His1 and its H-bonding Network
Article Snippet: The PCR product was gel purified (1% agarose) and treated with T4 DNA polymerase to create overhangs complementary to the Ligation Independent Cloning (LIC) vector pET30 Xa/LIC (Novagen™). .. The insert was annealed to the vector and NovaBlue (Novagen™) competent cells were transformed with the pET30 Xa/LIC plasmid containing the sodN mutant gene for selection. .. The plasmid containing mutagenic sodN was isolated using a plasmid mini prep kit (Qiagen) and DNA sequencing was performed to confirm the expected base sequence for the mutant (University of Massachusetts-Amherst DNA sequencing facility).

Article Title: The communication between the zinc and nickel sites in dimeric HypA: Metal recognition and pH sensing
Article Snippet: Point mutations of Cys residues in the two CxxC motifs associated with zinc binding and the two His residues (His79 and His95) that flank these two CxxC motifs were accomplished using the QuikChange (Stratagene™) mutagenesis kit and the pJI100 plasmid. .. The PCR product was used to transform into NovaBlue (Novagen™) competent cells.

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of His1 and its H-bonding Network
Article Snippet: The R47A-NiSOD mutant protein was prepared by PCR of WT-NiSOD pET-30 Xa/LIC plasmid with mutagenic 5’- and 3’-primers and the E17A/R47A-NiSOD and E17R/R47E-NiSOD double mutations were generated by PCR of the R47A-NiSOD pET-30 Xa/LIC plasmid with mutagenic 5’- and 3’-primers using the QuikChange® XL Site-Directed Mutagenesis Kit (supporting information ). .. NovaBlue (Novagen™) competent cells were transformed with the PCR product for mutant gene for selection.

Article Title: Characterization of a Novel Serine Protease Inhibitor Gene from a Marine Metagenome
Article Snippet: The PCR cycling consisted of denaturation steps (96 °C for 2 min), 30 cycles at 94 °C for 40 s, 58.5 °C for 30 s, and 72 °C for 2 min, as well as a final extension step at 72 °C for 10 min. After amplification, the PCR product mixture was digested by Bam HI as well as Hin dIII, and directly ligated into the pETBlue-2 (Novagen) expression vector cleaved with the same enzymes. .. The resulting recombinant plasmids were transferred into NovaBlue (Novagen) competent cells and placed on LB selection plates.

Article Title: Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms
Article Snippet: The PCR cycle consisted of an initial denaturation step of 96°C for 2 min, then 30 cycles of 94°C for 40 s, 57°C for 30 s, and 72°C for 2 min, followed by a final extension step at 72°C for 10 min. After amplification, the PCR product mixture was digested with Bam HI and Hin dIII, and ligated directly into pETBlue-2 (Novagen) expression vector cleaved with the same enzymes. .. The resultant recombinant plasmids were transferred into NovaBlue (Novagen) competent cells and plated onto LB selection plates.

Article Title: The Effects of Select Histidine to Cysteine Mutations on Transcriptional Regulation by E. coli RcnR
Article Snippet: Point mutations of the His60, His64 or His67 to Cys were made in the wild-type RcnR plasmid ( ) using QuikChange™ Site Directed Mutagenesis Kit (Stratagene). .. The PCR product was transformed into NovaBlue (Novagen) competent cells.

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of Cys2 and Cys6
Article Snippet: The PCR product was gel purified (1% agarose) and treated with T4 DNA polymerase to create overhangs complementary to the Ligation Independent Cloning (LIC) vector pET30 Xa/LIC (Novagen™ ). .. The insert was annealed to the vector and NovaBlue (Novagen™ ) competent cells were transformed with the pET30 Xa/LIC plasmid containing the sodN mutant gene for selection. .. The plasmid containing mutagenic sodN was isolated using plasmid mini prep kit from Qiagen and DNA sequencing was performed to confirm the expected base sequence for each mutant (University of Massachusetts-Amherst DNA sequencing facility).

Negative Control:

Article Title: Engineered Cpf1 variants with altered PAM specificities increase genome targeting range
Article Snippet: NovaBlue(DE3) E. coli (Novagen) were transformed with the variant library and plated on LB agar containing 25 μg/mL chloramphenicol. .. For each mutant PAM screened, the competent E. coli pool was transformed with 100 ng target plasmid containing the mutant PAM, incubated on ice for 15–30 min, heat shocked at 42 °C for 30s, and plated on LB agar (Affymetrix) containing 100 μg/mL ampicillin and 25 μg/mL chloramphenicol in the absence of IPTG.

Positron Emission Tomography:

Article Title: Concatenated Metallothionein as a Clonable Gold Label for Electron Microscopy
Article Snippet: The inserted MT gene was amplified from pET-3d-MT using the same forward primer as used for the pMAL-c2x-MT plasmid (above), and the reverse primer (5′-GTGACCACATGTCACAGCACGTGCACTTGTCC -3′), which contains an AflIII site at the MT-MT junction. .. The products of the ligation reactions for both constructs were transformed into Novablue (Novagen) E. coli cells.

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of His1 and its H-bonding Network
Article Snippet: The insert was annealed to the vector and NovaBlue (Novagen™) competent cells were transformed with the pET30 Xa/LIC plasmid containing the sodN mutant gene for selection. .. The plasmid containing mutagenic sodN was isolated using a plasmid mini prep kit (Qiagen) and DNA sequencing was performed to confirm the expected base sequence for the mutant (University of Massachusetts-Amherst DNA sequencing facility).

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of His1 and its H-bonding Network
Article Snippet: The R47A-NiSOD mutant protein was prepared by PCR of WT-NiSOD pET-30 Xa/LIC plasmid with mutagenic 5’- and 3’-primers and the E17A/R47A-NiSOD and E17R/R47E-NiSOD double mutations were generated by PCR of the R47A-NiSOD pET-30 Xa/LIC plasmid with mutagenic 5’- and 3’-primers using the QuikChange® XL Site-Directed Mutagenesis Kit (supporting information ). .. NovaBlue (Novagen™) competent cells were transformed with the PCR product for mutant gene for selection.

Produced:

Article Title: Concatenated Metallothionein as a Clonable Gold Label for Electron Microscopy
Article Snippet: Since NcoI and AflIII produced equivalent DNA ends, the second copy of MT was inserted with removal of both restriction sites at the ligation junction. .. The products of the ligation reactions for both constructs were transformed into Novablue (Novagen) E. coli cells.

Concentration Assay:

Article Title: Probing the Substrate Specificity and Protein-Protein Interactions of the E. coli Fatty Acid Dehydratase, FabA
Article Snippet: Polymerase chain reactions (PCR) were performed using a combination of varying DMSO and magnesium concentration and “step down” annealing temperatures. .. Digested PCR products were transformed into NovaBlue (EMD Millipore).

Recombinant:

Article Title: Enhancement of astaxanthin production in Xanthophyllomyces dendrorhous by efficient method for the complete deletion of genes
Article Snippet: This method has the potential to improve industrial production of various isoprenoids in X. dendrorhous without a decrease in cell growth. .. NovaBlue (Novagen, Madison, WI, USA) was used as the E. coli host strain for recombinant DNA manipulation. .. X. dendrorhous (NBRC 10129) was used as the parental host strain for gene expression.

Article Title: Development of a multi-gene expression system in Xanthophyllomyces dendrorhous
Article Snippet: Therefore, genetic engineering using the developed system to strongly express multiple target genes in a platform strain overproducing mevalonate has the potential to improve production of various value-added isoprenoids in X. dendrorhous . .. NovaBlue (Novagen, Madison, WI, USA) was used as the Escherichia coli host strain for recombinant DNA manipulation. .. Xanthophyllomyces dendrorhous (NBRC 10129) was used as the parental host strain for gene expression.

Article Title: Characterization of a Novel Serine Protease Inhibitor Gene from a Marine Metagenome
Article Snippet: The PCR cycling consisted of denaturation steps (96 °C for 2 min), 30 cycles at 94 °C for 40 s, 58.5 °C for 30 s, and 72 °C for 2 min, as well as a final extension step at 72 °C for 10 min. After amplification, the PCR product mixture was digested by Bam HI as well as Hin dIII, and directly ligated into the pETBlue-2 (Novagen) expression vector cleaved with the same enzymes. .. The resulting recombinant plasmids were transferred into NovaBlue (Novagen) competent cells and placed on LB selection plates. .. After overnight incubation at 37 °C, positive white colonies were picked for the isolation of the recombinant expression plasmid.

Article Title: Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms
Article Snippet: The PCR cycle consisted of an initial denaturation step of 96°C for 2 min, then 30 cycles of 94°C for 40 s, 57°C for 30 s, and 72°C for 2 min, followed by a final extension step at 72°C for 10 min. After amplification, the PCR product mixture was digested with Bam HI and Hin dIII, and ligated directly into pETBlue-2 (Novagen) expression vector cleaved with the same enzymes. .. The resultant recombinant plasmids were transferred into NovaBlue (Novagen) competent cells and plated onto LB selection plates. .. After overnight incubation at 37°C, positive white colonies were picked for isolation of the recombinant expression plasmid, which was subsequently introduced into E. coli BL21(DE3)pLysS (Novagen) to express the target protein.

Variant Assay:

Article Title: Engineered Cpf1 variants with altered PAM specificities increase genome targeting range
Article Snippet: The variant library was assembled by cassette mutagenesis, mini-prepped, pooled, and precipitated with isopropanol. .. NovaBlue(DE3) E. coli (Novagen) were transformed with the variant library and plated on LB agar containing 25 μg/mL chloramphenicol. .. Surviving colonies were scraped and cultured in ZymoBroth with 25 μg/mL chloramphenicol to an O.D. of 0.4–0.6 and made competent using a Mix & Go kit (Zymo).

Article Title: Nickel Superoxide Dismutase: Structural and Functional Roles of His1 and its H-bonding Network
Article Snippet: NovaBlue (Novagen™) competent cells were transformed with the PCR product for mutant gene for selection. .. The plasmid containing mutagenic sodN was isolated using a plasmid mini prep kit (Qiagen) and DNA sequencing was performed to confirm the expected base sequence for the mutant (W. M. Keck DNA facility at Yale University, New Haven, CT).

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    Millipore novablue
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